Evaluating Change of Marginal Bone Height with Cone-Beam Computed Tomography Following Surgical Treatment with Guided Tissue Regeneration (Bone Grafting) or Access Flap Alone: A Retrospective Study

Background and Objectives: This study aimed to evaluate the change of bone height following treatment of human intrabony defects with guided tissue regeneration (GTR) with bone grafting or access flap alone by cone-beam computed tomography (CBCT) scan. Materials and methods: This study was conducted as a retrospective longitudinal study. In this study, a total of 2281 teeth sites were included: the GTR group had 1210 sites, and the Flap group had 1071 sites. In the GTR group, demineralized freeze-dried bone (DFDBA) particles in combination with resorbable collagen membrane were used. No regenerative material was applied to the Flap group. CBCT images were taken twice at baseline and at least 2.5 months postoperatively. Bone heights were measured using software on CBCT images. Results: The bony change between the GTR and Flap groups was significantly different (p = 0.00001). Both males and females in the GTR group had smaller bone loss than in the Flap group. In age groups, significant differences of bony height between the GTR and Flap groups were observed in the subgroups consisting of those 29–45 and 46–53 years old. The non-smoking subjects in the GTR group had higher bone heights than those in the Flap group. In the absence of systemic disease and medicine, bone formation was higher in the GTR group than in the Flap group. In terms of oral position, the #14–17, #34–37, and #44–47 subgroups of the GTR group showed higher levels of bone heights than those of the Flap group. Conclusions. The results of this study indicated that the GTR procedure offers the additional benefit of higher bone heights than the Flap procedure does.


Introduction
The main objective of periodontal surgery is to contribute to the long-term preservation of the periodontium by facilitating plaque removal and infection control. Periodontal surgery is divided into open flap surgery and guided tissue regeneration (GTR). Open flap surgery aims to reconstruct the periodontal pocket by removing inflamed tissue and subgingival calculus. Open flap surgery provides the following: (1) accessibility for proper professional scaling and root planing; (2) establishing a gingival morphology that facilitates self-performed infection control; and (3) creating new attachment in the destructive site of the gingiva [1]. GTR is the regenerative procedure using bone graft material and barrier membranes [2,3]. GTR has the advantage of mechanical reinforcement of bony defects with bone graft material and membranes, thus providing space maintenance during healing periods. In the previous study, GTR has been shown to result in significantly more periodontal regeneration [4]. Sculean et al. and Stavropoulos et al. investigated that GTR had more increased clinical attachment level (CAL) gains compared with flap alone in clinical studies [5,6]. These approaches (GTR or open flap surgery), while offering tangible benefits, remain inconclusive. Few cases did enough to provide evidence of which procedure is better.
Demineralized freeze-dried bone (DFDBA) particles have been used in combination with barrier membrane in GTR, resulting in enhanced periodontal regeneration due to the osteoinductive ability of DFDBA to stimulate bone formation. DFDBA is associated with the amount of bone morphogenic proteins that remain after the demineralization process is completed [7,8]. A previous study demonstrated that probing pocket depth (PPD) and CAL gain were observed in sites treated with a combination of DFDBA and barrier membrane [9]. Similarly, Kiany and Moloudi reported favorable results after using DFDBA and barrier membrane [10].
A barrier membrane should have five characteristics to support ideal function: biocompatibility, space-maintenance ability, tissue integration, cell occlusiveness, and ease of manipulation [11]. Resorbable membrane has been used in GTR due to its biocompatibility [12]. However, resorbable membrane has weak physical properties. To maintain infrabony defect with bone graft material, resorbable membrane needs to be strengthened. A cross-linking procedure has been introduced that increases mechanical property and slows the degradation of resorbable membranes [13]. A cross-linking technique of resorbable membranes has two classified methods: physical and chemical methods. Physically cross-linked membranes were manufactured using ultraviolet (UV) irradiation or a dehydrothermal (DHT) technique. Chemically cross-linked membranes were made by glutaraldehyde (GTA) and hexamethylene diisocyanate (HMDI) [14]. A previous study demonstrated that cross-linked membranes overcome a shortage of non-cross-linked membranes [15].
To gain accurate results of periodontal treatment, various measurement methods have been developed. Clinical methods such as bone sounding are not always able to provide accurate tissue measurement. Surgical reentry and histologic evaluation are prohibited due to ethical issues. Therefore, radiographic methods (intraoral periapical and panoramic radiographs) were used to evaluate differences in marginal bone loss before and after surgery. The limitation of conventional radiographic measurement is that it cannot provide multi-dimensional assessment. Cone-beam computed tomography (CBCT), a reliable and widely used tool, provides three-dimensional exploration of periodontal osseous defects and assesses the furcation area of molars [16][17][18].
The aim of this study was to evaluate the change of bone height following treatment of human intrabony defects with GTR (bone grafting) or access flap alone.

Study Population
The study was conducted as a retrospective longitudinal study. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board for Clinical Research at Dankook University College of Dentistry Jukjeon Dental Hospital (approval no. 201910-001-002). From May 2015 to May 2018, 178 patients with 2520 sites (GTR: 97 patients (1296 sites); Flap: 81 patients (1224 sites)) who visited the department of periodontics at Dankook University College of dentistry Jukjeon Dental Hospital for periodontal treatment were selected. All patients met the inclusion criteria as follows by referring to previous studies [19][20][21].

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Clinical and radiographical diagnosis of severe chronic periodontitis; • Presence of at least one intrabony defect of ≥ 3 mm on the radiographs; • PPD ≥5 mm in the intrabony defects. Written informed consent was obtained from all the patients. Each site designated mesial and distal aspects on a panoramic view of CBCT images. After sorting due to scattering of CBCT image and extraction (28 teeth (56 sites) in Flap group) during the follow-up period, we divided the 2 groups by site (GTR: 1210 sites and Flap: 1071 sites). Premolars and molars of maxilla and mandible were included. Study participants were excluded if they did not complete periodontal treatment, had teeth extracted during the follow-up period, had no CBCT images both before and after treatment, and had <5 mm PPD, which is not suitable for periodontal surgery (GTR or Flap) ( Figure 1). Sample size calculation in this study referenced previous similar research by Tonetti et al. [20]. The power of 0.8 and an alpha level of 0.05 were used to detect the difference between groups.

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Presence of at least one intrabony defect of ≥ 3 mm on the radiographs; • PPD ≥5 mm in the intrabony defects.
Written informed consent was obtained from all the patients. Each site designated mesial and distal aspects on a panoramic view of CBCT images. After sorting due to scattering of CBCT image and extraction (28 teeth (56 sites) in Flap group) during the follow-up period, we divided the 2 groups by site (GTR: 1210 sites and Flap: 1071 sites). Premolars and molars of maxilla and mandible were included. Study participants were excluded if they did not complete periodontal treatment, had teeth extracted during the follow-up period, had no CBCT images both before and after treatment, and had <5mm PPD, which is not suitable for periodontal surgery (GTR or Flap) ( Figure 1). Sample size calculation in this study referenced previous similar research by Tonetti et al. [20]. The power of 0.8 and an alpha level of 0.05 were used to detect the difference between groups.

Pre-Treatment
Scaling or root planing was performed on all participants prior to the experimental phase.

Surgical Procedures
One periodontist (JTL) performed all surgical procedures. The patients received oral hygiene instructions before periodontal surgery. Under local anesthesia using 2% lidocaine (1:100,000 epinephrine, Huons Co., Ltd., Sungnam, Korea), the surgical sites were treated. In the GTR group, a full thickness flap was performed. The exposed defects were carefully scaled and root planed using a combination of mechanical and hand instruments. DFDBA (Human Cortical Powder, Demineralised, DIZG, Berlin, Germany) was applied to the defect area. A cross-linked-collagen membrane (OssGuide, Hyundai bioland Co., Ltd., Cheongju, Korea) composed of porcine pericardium-derived type I collagen was used. It was trimmed to the local anatomy and then positioned on the graft material. Then, the flap was replaced and sutured with resorbable sutures (4-0 Vicryl, Ethicon, Somerville, NJ, USA). All patients were prescribed amoxycillin and clavulanic acid 375 mg (Augmentin, Ilsung Pharma Co., Seoul, Korea), naproxen sodium (Anaprox, Jongeun Dang Pharmceutical, Co., Seoul, Korea), and almagate (Almagel, Yuhan Pharma

Pre-Treatment
Scaling or root planing was performed on all participants prior to the experimental phase.

Surgical Procedures
One periodontist (JTL) performed all surgical procedures. The patients received oral hygiene instructions before periodontal surgery. Under local anesthesia using 2% lidocaine (1:100,000 epinephrine, Huons Co., Ltd., Sungnam, Korea), the surgical sites were treated. In the GTR group, a full thickness flap was performed. The exposed defects were carefully scaled and root planed using a combination of mechanical and hand instruments. DFDBA (Human Cortical Powder, Demineralised, DIZG, Berlin, Germany) was applied to the defect area. A cross-linked-collagen membrane (OssGuide, Hyundai bioland Co., Ltd., Cheongju, Korea) composed of porcine pericardium-derived type I collagen was used. It was trimmed to the local anatomy and then positioned on the graft material. Then, the flap was replaced and sutured with resorbable sutures (4-0 Vicryl, Ethicon, Somerville, NJ, USA). All patients were prescribed amoxycillin and clavulanic acid 375 mg (Augmentin, Ilsung Pharma Co., Seoul, Korea), naproxen sodium (Anaprox, Jongeun Dang Pharmceutical, Co., Seoul, Korea), and almagate (Almagel, Yuhan Pharma Co., Seoul, Korea) 3 times daily for 1 week unless an allergy to penicillin was present. All patients were instructed to rinse for 30 seconds twice daily with 0.12% chlorhexidine gluconate (Hexamedin, Bukwang Pharmaceutical, Ansan, Korea) for 1 week. Sutures were removed 2 weeks post-surgery. In the Flap group, full thickness flap, scaling, and root planing were performed. Irregular bony protrusion was removed with rotary or hand instruments. After debridement, the flap was repositioned and sutured. The same medicines were prescribed as in the GTR group ( Figure 2). Co., Seoul, Korea) 3 times daily for 1 week unless an allergy to penicillin was present. All patients were instructed to rinse for 30 seconds twice daily with 0.12% chlorhexidine gluconate (Hexamedin, Bukwang Pharmaceutical, Ansan, Korea) for 1 week. Sutures were removed 2 weeks post-surgery. In the Flap group, full thickness flap, scaling, and root planing were performed. Irregular bony protrusion was removed with rotary or hand instruments. After debridement, the flap was repositioned and sutured. The same medicines were prescribed as in the GTR group ( Figure 2).

CBCT Taking and Maintenance Care (3 and 6 Months)
CBCT images were taken twice at baseline (before periodontal surgery) and at least 2.5 months postoperatively. A supportive care program and professional calculus removal were provided to all patients at 3 and 6 months. After that, regular oral cleaning was performed every 6 months if the patients maintained their oral hygiene.

Radiographic Evaluation and Bone Height Measurement
A CBCT scanner (Kodak 9500, Carestream Health, Rochester, NY, USA) was used in this study, providing a grayscale image of 14 bits with a voxel size of 0.2 mm per side. The

CBCT Taking and Maintenance Care (3 and 6 Months)
CBCT images were taken twice at baseline (before periodontal surgery) and at least 2.5 months postoperatively. A supportive care program and professional calculus removal were provided to all patients at 3 and 6 months. After that, regular oral cleaning was performed every 6 months if the patients maintained their oral hygiene.

Radiographic Evaluation and Bone Height Measurement
A CBCT scanner (Kodak 9500, Carestream Health, Rochester, NY, USA) was used in this study, providing a grayscale image of 14 bits with a voxel size of 0.2 mm per side. The CBCT images were viewed using 3D imaging software (OnDemand 3D, Cybermed Co., Seoul, Korea). In order to evaluate change of bone height, CBCT images were measured twice before and after treatment on the same site. The cemento-enamel junction (CEJ) was set as an unchanged reference [16]. If an implant was involved, the reference point was the connection between the abutment and the crown. In the preoperative CBCT image, the distance of the CEJ-base of alveolar bone of the mesial and distal sites of the teeth was measured ( Figure 3A-D). To overlap the images of the same cross-section as much as possible before and after treatment, imaginary lines were used ( Figure 3E; connecting line of CEJ, A, and D: bone height of first CBCT image, Figure 3F: connecting line of basic point between Figure 3A,D). The distance of the same site after treatment was measured on the second CBCT image ( Figure  cina 2021, 57, x FOR PEER REVIEW CBCT images were viewed using 3D imaging software (OnDemand 3D, Cyberm Seoul, Korea). In order to evaluate change of bone height, CBCT images were me twice before and after treatment on the same site. The cemento-enamel junction (CE set as an unchanged reference [16]. If an implant was involved, the reference poi the connection between the abutment and the crown. In the preoperative CBCT the distance of the CEJ-base of alveolar bone of the mesial and distal sites of the tee measured ( Figure 3A-D). To overlap the images of the same cross-section as m possible before and after treatment, imaginary lines were used ( Figure 3E; connecti of CEJ, A, and D: bone height of first CBCT image, Figure 3F: connecting line of basi between Figure 3A,D). The distance of the same site after treatment was measured second CBCT image ( Figure

Statistical Analysis
Statistical analysis was performed using SPSS software (SPSS version 23.0, C IL, USA). To compare variables between GTR and Flap groups, a Mann-Whitney was used. Kruskal-Wallis was used to compare variables in 3 or more groups inc smoking, systemic disease, medicine, oral regions, and precise site. The thresh statistical significance was 5%. The post hoc Bonferroni correction was used for m comparisons between groups. A logistic regression model was used for multiv

Statistical Analysis
Statistical analysis was performed using SPSS software (SPSS version 23.0, Chicago, IL, USA). To compare variables between GTR and Flap groups, a Mann-Whitney U test was used. Kruskal-Wallis was used to compare variables in 3 or more groups including smoking, systemic disease, medicine, oral regions, and precise site. The threshold for statistical significance was 5%. The post hoc Bonferroni correction was used for multiple comparisons between groups. A logistic regression model was used for multivariable analysis.

Association of Tooth Site and Bone Height between GTR and Flap
The bony height values of #14D, #15M, and #47M in the GTR group were significantly higher than those in the Flap group (p = 0.016, p = 0.036, and p = 0.023, for each). In addition, the bony height values of #14M, #17D, and #35M in the GTR group were close to significance (p = 0.068, p = 0.060, and p = 0.073, respectively). However, the change of bony height value for #25D in the Flap group revealed a significant difference to that in the GTR group (p = 0.043) ( Table 2).

Association of Sex and Bone Height between GTR and Flap
In the GTR group, women were more effective at #14D and #15M (p = 0.028 and p = 0.009, for each). In males, GTR increased bone resorption at #15D. In males, the GTR group had increased bone resorption at #15D compared with the Flap group (0.54 ± 1.32; GTR vs. 0.25 ± 0.85; Flap, p = 0.635). In the case of #24D, the amount of bony change of GTR group was smaller than that of the Flap group regardless of gender (Male: 0.11 ± 0.77; GTR vs. −0. 15 Table 3).

Association of Smoking and Bone Height between GTR and Flap
The GTR and Flap groups differed by smoking status. There was a significant difference between the two groups in non-smoking (p = 0.009). Among smokers, 10 of 32 sites in the GTR group showed less bone healing than in the Flap group (#14M, #16D, #24M, #24D, #25M, #27M, #27D, #34M, #44D, and #46D). In non-smokers, the bone healing of the #24M and #34 sites was better in the GTR group (p = 0.011 and p = 0.019, respectively) ( Table 4).

Association of Age and Bone Height between GTR and Flap
At 29-45 years of age, 24 sites showed better bone recovery in the GTR group than in the Flap group. Four sites (#14D, #35M, #47M, and #47D) in the GTR group showed significant differences (p = 0.016, p = 0.036, p = 0.010, and p = 0.011, for each). The 46-53 subgroup showed less bone resorption and bone filling in the GTR group than in the Flap group. In the 54-76 subgroup, the number of favorable healing sites in the GTR group was less than in the sites of the other two subgroups. No difference was found in the 54-76 subgroup (Table 5).

Association of CBCT Interval and Bone Height between GTR and Flap
The smaller the interval between before and after CBCT, the more bone increase and the less bone resorption. In particular, in the 860-1543 days group, there was a significant difference in bone growth or small bone resorption at #14D, #26M, and #35M (p = 0.048, p = 0.032, and p = 0.042, respectively) ( Table 6).

Discussion
The present study showed that the change of bone height after GTR and after access flap alone procedures showed differences. Our findings indicate that the bone height of several teeth in the GTR group was significantly higher compared with those teeth in the Flap group (#14D, #15M, and #47M). Females in the GTR group had significantly more favorable values than males. In males, #47 showed significant healing of bone height. Regarding smoking, there was notable bone loss in the GTR group among smokers. All patients were divided by three age-related subgroups consisting of those 29-45, 46-53, and 54-76 years old. At lower ages (29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45), the GTR group showed less bone resorption than the Flap group. The smaller the interval between before and after CBCT, the more bone increase and the less bone resorption. It was observed that the smaller the interval between shots of CBCT, the less bone resorption. In particular, in the absence of systemic diseases or no medications, the GTR group showed less bone resorption and better healing than the Flap group. A multivariable analysis suggested that the type of surgery and surgical site (upper/lower jaw) were associated with bone resorption after treatment.
In this study, the comparison between groups showed less alveolar bone loss and superior bone healing in the GTR group vs. the Flap group as a whole. The same result was seen in a previous study. Tonetti et al. suggested that regenerative periodontal surgery with GTR had PPD reduction and CAL gain [20]. They also noted that the absolute value of the added healing portion was relatively small, but it was in agreement with other similar research studies [22,23]. In a systemic review of 13 articles, regenerative surgeries have shown an adjunctive benefit of CAL gain [24]. A distinctive point observed in this study was that 28 teeth were extracted only from the Flap group during the follow-up period after periodontal surgery. It seems that the GTR procedure is a superior treatment to prolong tooth life span compared with the Flap procedure. Long-term study of GTR vs. open flap reported that there was no significant difference of bone loss between GTR and open Flap surgery [21]. The present study revealed that GTR in the mandible yielded better bone gain than in the maxilla. These outcomes are similar to those reported by Odontuya et al. [25]. Other researchers explained that this difference was due to the complex morphology and accessibility of molars such as root cuvature and furcations [26,27].
This study used DFDBA in combination with resorbable membrane in the GTR group. DFDBA is an allograft with potential osteoinductive ability to expose BMPs that promote osteoblast differentiation, and it would be beneficial for bone regeneration [28,29]. Camelo et al. demonstrated that DFDBA with collagen membrane had better clinical outcomes of bony healing in a human study [30]. In an animal study, the combination of membrane and DFDBA has been proven to regenerate bone [31]. By demineralized processing, DFDBA has higher osteoinductivity but lower mechanical property. Bone graft material should prevent the resorbable membrane from moving downward. For this reason, physical strength is necessary. DFDBA has relatively weaker mechanical properties than freeze-dried bone allograft (FDBA) or xenograft, and it might affect results when used clinically. A previous study failed to find a difference between bone grafting with DFDBA and a no-bone grafting procedure [32].
Resorbable membranes have been introduced as a barrier to prevent moving epithelium apically [33]. While resorbable membranes have many favorable aspects, they have major limitations, including weaker physical strength [34]. A previous study found that it takes approximately 4-24 weeks for the degradation of collagen membranes [11]. Crosslinked membranes have been developed to increase the stiffness and delay the degradation of resorbable membranes and have shown superior physical strength relative to non-crosslinked membranes [13,35]. Cross-linked membranes were used in this study. In a previous study, the application of resorbable membranes was favorable at infrabony defects [32]. Another study demonstrated that combining bone graft material with resorbable membrane resulted in more benefit than only membrane [36]. However, Stavropoulos et al. did not find any difference between those two methods (bone + membrane vs. membrane only) [6]. In this study, porcine collagen was used as the source of resorbable membranes. Bovine collagen is one of the major sources of resorbable membranes, but the drawback of this collagen is the associated allergic reactions [37]. Porcine collagen in a known alternative to bovine collagen. A previous study found that porcine collagen led to minimal allergic reactions because porcine collagen seemed more similar to human collagen [38]. Lee et al. reported a similar clinical expediency and properties when comparing porcine cross-linked vs. non-cross-linked collagen membrane [39].
This study differs from previous studies in that it compared the situation before and after treatment using CBCT. The detection of periodontal bone loss is mandatory for accurate diagnosis. Clinical methods including probing have shown limitations in reliability. Various factors such as the probing force, shape of the tip, and direction of the probe affect the clinical results. In addition, it is not easy to accurately assess the healing point due to resistance of tissue [40]. It is difficult to get an accurate image using overlapping images from conventional radiographs such as intraoral periapical and panoramic radiographs. CBCT has been introduced to overcome the drawbacks of conventional radiographic methods. CBCT can get a specific cross-sectional image and can reconstruct radiographic images with a multi-dimensional view [16][17][18]. There were several previous studies in which CBCT was used to measure the amount of bone loss in periodontitis. Mohan et al. reported that there was no difference in bone loss between CBCT and actual measurement of surgically exposed osseous defects [40]. In addition, CBCT was useful for identifying buccal and lingual bony defects in aggressive periodontitis. Another study has concluded that CBCT had the ability to assess the maxillary or mandibular furcation area [41]. CBCT can identify the root concavities of premolars and bone loss pattern [42]. An important advantage of CBCT is the low radiation dose. The radiation of CBCT has been reported to be 15 times lower than conventional radiography [43].
The limitation of this restorative study is that the conditions such as systemic disease and oral environment of the test and control group could not be perfectly matched. To enhance these drawback, future studies will investigate prospective research for the controlled subjects. In addition, other bone substitutes including xenograft and alloplast might be suggested to use for further research. A strength of the present study is that a large number of subjects using CBCT were involved. The design of this study differs from previous studies in that it directly observed bone changes using CBCT. PPD and CAL may be good clinical indicators. However, radiologic measurement with CBCT is also good and accurate for evaluating the effectiveness of periodontal treatment.

Conclusions
Despite the limitations of this study, the results suggest that GTR with bone grafting can be a more effective method for bone healing and delay the extraction of teeth than Flap alone.